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Current Practice in Bicistronic IRES Reporter Use: A Systematic Review.

Guus Gijsbertus Hubert van den Akker¹, Federico Zacchini^2,3, Bas Adrianus Catharina Housmans¹

¹Department of Orthopedic Surgery, Maastricht University, Medical Center+, 6229 ER Maastricht, The Netherlands.

International Journal of Molecular Sciences

|June 2, 2021

Summary

Bicistronic reporter assays are widely used but show methodological variability. This review highlights inconsistencies in reporting and suggests best practices for reproducible internal ribosomal entry site (IRES) research.

Keywords:

IRES bicistronic reporter dicistronic reporter mRNA translation ribosome systematic review

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Area of Science:

Molecular Biology
Genetics
Biochemistry

Background:

Bicistronic reporter assays are crucial for studying gene expression, internal ribosomal entry site (IRES) function, and cap-independent translational elements (CITE).
Significant methodological variability exists in the application and reporting of these assays.
This inconsistency hinders data reproducibility and interpretation in the field.

Purpose of the Study:

To systematically review and analyze the methodological details, data presentation, and normalization procedures in published bicistronic IRES reporter studies.
To identify common practices and inconsistencies in experimental design and data reporting.
To provide recommendations for improving transparency and reproducibility.

Main Methods:

A systematic literature search identified 257 bicistronic IRES reporter studies (1994-2020).
176 full-text articles were analyzed for details on reporter design, cell systems, transfection, and analysis time points.
Cell-free translation assays were evaluated for in vitro transcription, lysate type, incubation, and temperature.

Main Results:

Analysis revealed considerable variability in experimental parameters and data reporting across studies.
Inconsistent reporting of bicistronic reporter design, cell types, and assay conditions was observed.
Diverse methods for data presentation, normalization, and control experiments were identified, complicating comparisons.

Conclusions:

Inconsistencies in bicistronic reporter assay methodology and data presentation impede reproducibility.
Adopting standardized reporting practices and clear methodological descriptions is essential.
Implementing these recommendations will enhance transparency and facilitate reliable data replication in IRES research.